Field of the Invention
The present invention relates to: an isolated T cell receptor (hereinafter referred to as “TCR”) capable of recognizing a cancer antigen restricted to HLA-A*24:02; and the uses of the TCR for detection of a cancer antigen restricted to HLA-A*24:02, for cancer treatment, and for other purposes.
Related Background Art
In Japan, approximately 350,000 people die of cancers every year, and approximately 700,000 are newly diagnosed as cancers. In the cancer treatment, mainly three standard treatments are given: surgery, anticancer drug, and radiation therapy. When no improvement is observed after such treatments, there is no choice but palliative care for relieving various cancer-caused pains. In such cancer treatment situations in Japan, patients themselves or families of the patients are called “cancer refugees” from their endeavor searching for further treatments, bringing about a social problem.
Currently, cancer vaccination is actively studied as an intermediate treatment between the standard treatment and the palliative care. The cancer vaccination is a treatment given in order to eliminate or regress cancer by activating the immunity of the patients themselves. Provenge (Sipuleucel-T) approved by the United State Food and Drug Administration (FDA) in April, 2010, is one used in the cancer vaccination. Provenge is approved for patients with prostate cancer that is metastatic and hormonal therapy resistant. In the treatment method with Provenge, peripheral blood of the patient is stimulated using a PAP (prostatic-acid phosphatase) protein over-expressed in most cases of prostate cancer, and then Provenge is transferred into the body of the patient. Whether the cancer vaccination succeeds or not depends on the immunity of the patients themselves. For this reason, it is believed that the cancer vaccination is more effective if performed before the symptom of the patient is worsened by various standard treatments. In the future, such a cancer vaccination is desirably approved as a treatment choice in place of the anticancer drug treatment or as a treatment method applicable in combination with an anticancer drug.
The cancer vaccination is roughly classified into two: an antigen non-specific treatment method and an antigen specific treatment method. An antigen non-specific immunotherapy has started since 1970s using picibanil, Krestin, killed Mycobacterium tuberculosis (BCG-CWS), or the like having an immunostimulating action. In 1980s, immunotherapies were performed using cytokines such as IL-2, and LAK (lymphokine activated killer cell) therapies were performed in which T cells non-specifically proliferated were returned into the body of the patient. However, any of these failed to clearly produce clinical effects, and are not covered by health insurance.
It was reported in 1991 that cytotoxic T lymphocytes (hereinafter referred to as “CTLs”), which were separated from a patient and proliferated, specifically recognized cancer antigens called MAGE (melanoma antigen) expressed at high level on cancer cells, and killed the cancer cells (Science 1991. 254: 1643-1647). Moreover, in 1994, there was a report on a mechanism of CTLs to recognize and kill peptide fragments presented by MHC (Major Histocompatibility Complex, called HLA in human) on cancer cell surfaces (Science 1994; 264: 716-719). After that, researchers all over the world identified various peptide fragments presented by HLA. In 1996, Altman et al. developed MHC tetramer reagents capable of detecting specific CTLs (Science, 1996; 274: 94-96). This made it possible to directly demonstrate CTL detection which has been so far verified only indirectly by 51Cr release assay. The melanoma-targeting cancer peptide vaccination reported in 1998 produced surprising clinical outcome, and thus cancer immunotherapy has flourished at once (Nat Med, 1998; 4: 321-327). Since then, cancer peptide vaccinations through subcutaneous or intradermal inoculation with peptides have been in progress on a worldwide scale.
In 2004, Rosenberg et al. reported that the response rate by cancer vaccination was lower than expected, and cautioned that the subjects of the clinical trials were terminal cancer patients (Nat Med, 2004; 10: 909-915). This is because whether or not CTLs that attack cancer cells in the body of the patients after inoculation with a peptide vaccine are proliferated as hoped depends on the immunity of the patients themselves. Hence, it seems natural that if the immunity is greatly lowered due to chemotherapy or radiation therapy, the response rate is also lowered. Patients inoculated with a peptide vaccine require steps of: activating T cells by causing dendritic cells or macrophages, which are called antigen-presenting cells, residing in the vicinity of the inoculation site to incorporate the peptide and present it to MHC on the cell surface; and further activating T cells by recruiting antigen-presenting cells from regional lymph nodes also. In patients with the immunity greatly lowered due to an anticancer drug or radiation therapy, presumably the number of antigen-presenting cells is reduced, and that the antigen-presenting cells do not function properly. This may limit the anticancer effect. For this reason, a dendritic cell treatment is performed: such antigen-presenting cells as dendritic cells or macrophages are separated and cultured to proliferate in vitro, and patients are inoculated with the antigen-presenting cells ready to present the antigen. However, even if such a treatment is performed, the effect is thought to be limited in some cases as in peptide vaccination because patients having the immunity lowered do not have a sufficient amount of T cells in the first place, or because the T cells are not activated sufficiently.
Then, in order to compensate for the shortcomings of the peptide vaccine and the dendritic cell treatment, a CTL treatment is performed in which T cells activated in vitro are returned into the body of the patient. For this method, a dedicated cell processing center (CPC) is required to transfer the cells into the blood. Since complicated cell-culturing operations are repeated, developments of, for example, automated culture system and the like are accordingly in progress. However, quite difficult culture techniques are required regarding how specific CTLs are proliferated in vitro efficiently within a short period of time.
T cells act by recognizing and binding to a complex of a MHC molecule and an antigenic peptide (hereinafter referred to as “MHC/peptide complex”) presented on the cell surface of targets such as antigen-presenting cells, cancer cells, and infected cells, via a TCR expressed on the surface of the T cells. It is believed that CD8 positive T cells bind only to a MHC class I molecule/antigenic peptide complex (MHC class I restriction), and that CD4 positive T cells bind only to a MHC class II molecule/antigenic peptide complex (MHC class II restriction). While MHC class I molecules are expressed on most of nucleated cells and platelets, MHC class II molecules are expressed only on limited types of cells. For this reason, CD4 positive T cells can bind to dendritic cells, B cells, activating T cells, and the like expressing MHC class II molecules, but cannot directly binds to others such as tumor cells and infected cells. Nevertheless, it has been demonstrated that when a TCR gene derived from CD8 positive T cells (CTLs) believed to be restricted to MHC class I is introduced by genetic manipulation into CD4 positive CD8 negative T cells believed to be restricted to MHC class II, the T cells are activated by reacting in a CD8-molecule independent manner with antigen-presenting cells pulsed with a corresponding antigen, demonstrating a cytotoxic activity. Moreover, it has been reported that cancer can be specifically regressed by introducing a TCR gene derived from cancer antigen specific CTL into peripheral blood lymphocytes having a non-specific anti-tumor activity (J. Immunol., 2003; 171: 3287-3295). Accordingly, there have been active attempts to conduct TCR gene therapy in which TCR genes isolated from specific CTLs are artificially expressed in peripheral lymphocytes, and artificial CTLs are prepared to be returned into the body of the patient (Science 2006; 314: 126-129).
For approximately 20 years, quite a large number of peptide fragments (CTL epitopes) were identified from proteins associated with cancers, infectious diseases, autoimmune diseases, and engraftment. Even if CTL epitopes are derived from the same protein, the type of presented peptide fragments varies, depending on the HLA type. This is generally called HLA restriction of CTL epitope.
In a cancer vaccination, selection of a cancer antigen from which a CTL epitope is derived is a very important point for the target patient. A CTL epitope is desirably restricted to an HLA type widely possessed within human races, among extremely diverse HLA types. For example, while 1527 proteins are registered for HLA-A, there are 21 genotypes for the HLA-A type with a frequency of 0.1% or higher according to the paper which analyzed the HLA type of 1018 Japanese (Immunogenetics, 2005; 57: 717-729). Among these, HLA-A*24:02 was detected at a frequency of 36.2%, which suggests that 60 to 70% of Japanese have the HLA type. Accordingly, it is very meaningful to identify a CTL epitope restricted to an HLA type with such a high frequency.
While a great number of CTL epitopes have been reported, the result of evaluating 75 CTL epitopes specific to cancer antigen on the basis of nine criteria was reported in 2009 (Clin. Cancer Res., 2009; 15: 5323-5337). The nine criteria include (a) therapeutic function, (b) immunogenicity, (c) role of the antigen in oncogenicity, (d) specificity, (e) expression level and percent of antigen-positive cells, (f) stem cell expression, (g) number of patients with antigen-positive cancers, (h) number of antigenic epitopes, and (i) cellular location of antigen expression. According to this report, WT1 is the most promising CTL epitope in the cancer vaccination among the 75 cancer antigens.
Regarding what cancer antigen a vaccination to a target patient should target, it is desirable to carry out a diagnosis before the vaccination is started. For example, in performing HLA-A*24:02-restricted WT1 peptide vaccination, it is a prerequisite that the HLA type of the target patient should be HLA-A*24:02. The HLA type examination is an essential examination for organ transplantation or bone marrow transplantation, and is conducted in various facilities. In addition, since it is essential that the WT1 peptide restricted to HLA-A*24:02 should be presented on the target cancer cell surface to be recognized by CTLs, this condition is most desirably proved in order to perform the HLA-A*24:02-restricted WT1 peptide vaccination. Nevertheless, currently, no examination method is available for directly proving the condition; instead, an indirect examination is conducted. For example, utilized for WT1 are: a method in which the level of WT1 mRNA expressed is checked by quantitative PCR using a biopsy sample, a method in which an expression of a WT1 protein in cells is checked using an anti-WT1 antibody, and a method in which an HLA expression is checked using an anti-HLA antibody. Tools which have been studied for the direct proving include antibodies for specifically recognizing a state where a peptide binds to MHC (hereinafter referred to as “anti-pMHC antibody”), TCR tetramers utilizing TCR, scTCR (single chain TCR) multimers utilizing single chain TCR, and the like.
Regarding the anti-pMHC antibody, there were a report in 1989 on an antibody specifically recognizing MHC class II presenting a peptide (Nature, 1989; 338: 765-768), and a report in 1997 on an antibody recognizing a state where an OVA (ovalbumin)-derived peptide is presented by a mouse MHC class I molecule H-2Kb (Immunity, 1997; 6: 715-726). Many researchers have started attempting to obtain similar antibodies.
Molecules constituting a MHC/peptide complex are in such a state that a MHC having a molecular weight of approximately 45 kDa is non-covalently bonded to β2m (β2-microglobulin) of approximately 12 kDa. For example, in a case of a peptide presented by MHC class I, approximately 8 to 11 amino acids are bonded. It is very difficult to obtain, by hybridoma technology, antibodies specifically recognizing only the difference in presented peptides. In fact, only several antibodies were reported in the past 20 years.
For example, there was a report in 1996 that an antibody against a MHC/peptide complex was obtained by the phage display technology using a library derived from an immunized mouse (PNAS, 1996; 93: 1820-1824). Then, there was a report in 2000 that an antibody specific to an HLA-A1/MAGE-A1 complex was obtained from a library derived from non-immunized human (PNAS, 2000; 97: 7969-7974). Antibodies obtained in this manner can exhibit a reactivity when artificially bound to a peptide on the cell surface. However, it is known to be very difficult to develop an antibody capable of exhibiting a reactivity when an endogenous peptide is presented in a natural state on the cell surface. CTLs are believed to demonstrate a cytotoxic activity when activated by specifically binding to approximately several to 10 MHC/peptide complexes that the CTLs can specifically recognize among several tens of thousands of MHC/peptide complexes expressed on the surface of one target cell. In order to detect this phenomenon without using specific CTLs, a reagent similarly capable of binding to approximately several to 10 MHC/peptide complexes per cell has to be developed and detected with an analyzer. Even in a case for example where proteins expressed on the cell surface are analyzed by flow cytometry using a specific antibody, if less than 100 target proteins are expressed per cell, it is difficult to determine from the analysis result that the molecules of interest are expressed in many cases. It is pointed out that the currently-available analyzers may not have an enough detection sensitivity.
Heretofore, a MHC/peptide complex detection system utilizing soluble TCR has been conceived (J Mol Biol, 1994; 242: 655-669). However, its applicability has been considered to be low because it is difficult to obtain α-chain and β-chain proteins constituting the TCR while their conformations as a functional recombinant protein complex are being kept, and because the binding strength is weak between the TCR and the MHC/peptide complex. Nevertheless, as in the case of MHC tetramer reagent synthesis technique, it was reported in 2004 that such a TCR was practically usable in the form of tetramer (Nat Biotechnol, 2004; 22: 1429-1434). In 2006, a reagent capable of specifically detecting a MHC/peptide complex was successfully produced by modifying TCR proteins forming a heterodimer of α-chain and β-chain into a single chain TCR (scTCR) having Vα, Vβ, and Cβ linked to each other, followed by multimerization (J. Immunol. 2006; 176: 3223-3232).